Cloning and Molecular Characterization of CcNRT2.1/CcNAR2, a Putative Inducible High Affinity Nitrate Transport System in Capsicum chinense Jacq. Roots

Abstract

NRT2 family of nitrate transporters normally require a partner protein, NAR2 (nitrate assimilation related protein), to transport nitrate to a high-affinity (high affinity nitrate transport system, HATS), although its role is still not well understood. In this study, the CcNRT2.1 and CcNAR2 cDNAs from of Capsicum chinense were cloned and characterized in terms of their structure, phylogeny, and their organ-specific transcriptional regulation, and by ligh/dark cycle, N and sucrose. CcNRT2.1 (putative molecular mass 57.5 kDa) and CcNAR2 (22.66 kDa) retained the characteristic domains of NRT2 and NAR2 family, respectively. CcNRT2.1 is expressed exclusively in the root, and both western blot and protein immunolocalization, demonstrated its exclusive location in root epidermal and cortical cells. CcNAR2 is expressed mainly in the root, but also in fruits of 25 days post-anthesis (DPA). The presence of two protein bands (~60 and ~80 kDa) detected in the western blot, suggested that CcNRT2.1 monomer and CcNRT2.1/CcNAR2 heterodimer work together for nitrate transport function in roots. Both genes were induced by NO3−, NH4+ and glutamate, and CcNRT2.1 was induced by sucrose. The co-expression of both genes in the specific zone of the root (2–4 cm from the apex) in which the high-affinity nitrate transport occurs (evaluated by root NO3− endogenous content, extracellular pH alkalinization, and H+ net flux by MIFE technique), strongly supports that CcNRT2.1/CcNAR2 belong to a HATS, with a functional similarity to AtNRT2.1/AtNAR2.1 of Arabidopsis. These are the first components of a HATS characterized in the Capsicum genus.